Food Chemistry 133 (2012) 13071311

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Rapid Communication

Rising atmospheric CO2 concentration affects mineral nutrient and protein

concentration of wheat grain
Nimesha Fernando a, Joe Panozzo b, Michael Tausz c, Robert Norton a,d, Glenn Fitzgerald b,
Saman Seneweera a,
a
Department of Agriculture and Food Systems, Melbourne School of Land and Environment, The University of Melbourne, Natimuk Road, Private Box 260, Horsham,
Victoria 3401, Australia
b
Department of Primary Industries, Natimuk Road, Private Box 260, Horsham, Victoria 3401, Australia
c
Department of Forest and Ecosystem Science, Melbourne School of Land and Environment, The University of Melbourne, Water Street, Creswick, Victoria 3363, Australia
d
International Plant Nutrition Institute, 54 Florence Street, Horsham, Victoria 3400, Australia

a r t i c l e

i n f o

Article history:
Available online 8 February 2012
Keywords:
Free-Air Carbon dioxide Enrichment (FACE)
Iron
Grain mineral composition
Protein
Zinc

a b s t r a c t
Wheat (Triticum aestivum L. cv. Yitpi) was grown in the Australian Grains Free-Air Carbon dioxide Enrichment (AGFACE) facility under current ambient [CO2] (384 lmol mol1) and elevated [CO2]
(550 lmol mol1) in combination with two different times of sowing (TOS) to investigate the interactive
effect of [CO2] and grain lling conditions on wheat grain quality. Grains were sampled at harvest maturity and their protein and mineral nutrient (Ca, S, Zn and Fe) concentrations were measured. Protein concentration of the grain was decreased by 12.7% at elevated [CO2] and the largest reduction in grain
protein was observed at the later TOS (TOS2). Concentration of grain S, Ca, Fe and Zn were also signicantly decreased at elevated [CO2]. Most of the grain mineral nutrient concentrations were signicantly
increased at the TOS2 suggesting that rising temperature together with increased water stress are likely
to offset some of the negative effects of elevated [CO2] on grain mineral concentrations.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
Atmospheric carbon dioxide concentration [CO2] is expected to
reach 550 lmol mol1 by the middle of this century (Carter, Jones,
& Lu, 2007). Simultaneously, global temperature is predicted to rise
by an average of 1.54.5 C with more frequent occurrences of extreme climatic events such as heat waves and/or droughts (Carter
et al., 2007). Changes in these environmental variables directly or
indirectly affect plant growth, development, grain yield and quality
(Ainsworth & Rogers, 2007; Loladze, 2002; Seneweera, Makino,
Mae, & Basra, 2005).
Grain yield for C3 crops is expected to increase over the next
100 years due to increasing carbon dioxide concentration [CO2]
(Kimball et al., 2001), but this may occur at the cost of reducing
grain quality traits such as protein, mineral nutrient and starch
properties (Hgy et al., 2009; Rogers et al., 1998; Seneweera
et al., 1996). In particular, reduction of N concentration in grains
has been well documented under elevated carbon dioxide concentration [eCO2] (Erbs et al., 2010; Hgy et al., 2009; Kimball et al.,
2001). Other climatic factors such as high temperatures and soil
water stress are likely to have a negative impact on wheat grain
Corresponding author. Tel.: +61 3 5362 2164, mobile: +61 40187 9853; fax: +61
3 5362 0782.
E-mail address: samans@unimelb.edu.au (S. Seneweera).
0308-8146/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2012.01.105

protein (Corbellini, 1998; Panozzo & Eagles, 2000). Grain protein

concentration and its quality determine the functional properties
of the our (Shewry & Halford, 2002). Further, our protein concentration and composition is well associated with bread quality
and determines the price of bread wheat (Panozzo & Eagles,
2000; Shewry & Halford, 2002).
The lack of essential micronutrients in the diet, such as iron (Fe)
and zinc (Zn) is a growing global concern, and a major cause of
child mortality and mental impairment (World Health Organization, 2007). Currently, more than 2 billion people worldwide suffer
from Fe and Zn deciency (World Health Organization, 2007).
Every year, an estimated 400,000 children, predominantly in Asia,
die from Zn deciency, particularly in the regions where wheat is
the staple food (Cakmak, Pfeiffer, & McClafferty, 2010). Iron deciency is the most common cause of anaemia, and is also associated with mental and psychomotor development in children
(Cakmak et al., 2010). It has been suggested that rising [CO2] has
a major impact on cereal grain macro and micro nutrient concentrations (Erbs et al., 2010; Hgy et al., 2009; Kimball et al., 2001;
Seneweera et al., 1996). However, there is only a limited understanding of the impact of [eCO2] at high grain lling temperature
on grain micro nutrient concentrations under.
All the previous Free-Air Carbon dioxide Enrichment (FACE)
studies were conducted in high yielding conditions of either a humid climate or under regular water supply. No studies have been

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N. Fernando et al. / Food Chemistry 133 (2012) 13071311

conducted to investigate the impact of [eCO2] and high temperature during grain lling under low rainfall conditions. In this
experiment, we tested the hypothesis that [eCO2] further modies
the wheat grain quality in drier and warmer grain lling conditions. Delayed sowing treatment was introduced to mimic high
temperature, but other stress factors such as drought and vapour
pressure decit are also more common. For the rst time, we report the effects of [eCO2] and delayed sowing on the grain protein
and mineral concentration of wheat, using the FACE facility. The
ndings of this experiment will provide much needed information
on how climate change impacts on wheat grain quality.
2. Materials and methods
2.1. CO2 exposure and experiment conditions
The experiment was conducted during the growing seasons of
2008 and 2009 at the Department of Primary Industries Research
Station, Walpeup, Victoria, Australia (35120 S, 142000 E).
The experimental design was a completely randomized block
design. Wheat (Triticum aestivum L. cv. Yitpi) was grown in eight
[aCO2] rings (384 lmol CO2 mol1, 4 m diameter) and eight
[eCO2] rings (550 lmol CO2 mol1, 4 m diameter), completely randomized with two different sowing times: normal or TOS1 (late
May or early June) and late or TOS2 (mid or late June). The four
combinations of two levels of [CO2] and two sowing times were
allocated to the 16 rings with four replicates. Each ring consisted
of two plots (4 m long  1.8 m width) and the plots were sown
in eight rows of 0.21 m space between rows and 0.1 m space within rows.
[CO2] in the FACE rings (4 m diameter) was maintained at
550 lmol CO2 mol1 by injecting pure CO2 into the air from an
octagonal FACE ring (Mollah, Norton, & Huzzey, 2009). The ambient plot had no CO2 injection, where seasonal long average [CO2]
was 384 lmol CO2 mol1. The main soil type dominating in the
study site was Calcarosol according to the Australian Soil Classication Standards. Some of the growth conditions of TOS1 and
TOS2 in both growing seasons are described in Table 1. All agronomic practices in this experiment were similar to normal farming
practices of the region and fertilizer nitrogen supply was at a rate
of 50 kg/ha which is considered to be adequate for prevailing
growing conditions. Accumulated growing degree days (GDD)
were calculated by summing daily degree days according to Darroch and Baker (1990). Daily degree days were calculated as
Tn = (Tmax + Tmin)/2  Tb, where Tmax and Tmin are the maximum
and minimum daily temperatures, respectively, and Tb is the base
temperature (5 C).
2.2. Grain sample preparation
Ears harvested at maturity were dried at room temperature and
grains were separated and aspirated (Vacuum separator, Kimseed,
Australia) to remove the remaining husk and dust, and stored at

20 C in plastic containers to avoid moisture absorption until further analyses.

2.3. Grain protein analysis
Total protein content in the whole-grain was determined by
Near Infrared Reectance Spectroscopy (NIR, Foss, Sweden) (AACC
method 39-25) and is expressed on a grain dry weight basis.
2.4. Grain Ca, S, Zn and Fe analysis
Grain samples were analysed for iron (Fe), zinc (Zn), calcium
(Ca) and sulphur (S) by inductively-coupled plasma atomic emission spectrometry (ICP-AMS) (Applied Research Laboratories,
3580B, Switzerland) after digestion in concentrated nitric acid
(HNO3) following the procedure of Zarcinas, Cartwright, and Spencer (1987). Briey, 0.8 g of ground wheat grains were cold digested
with ultrapure concentrated HNO3 (9 ml) over night in tubes before heating. Hydrochloric acid (HCl) was added during two stages
of digestion (0.4 ml + 0.2 ml nish) under a controlled regime in an
open vessel (75 ml borosilicate glass tube), and the nal volume
was made up to 25 ml. Generally, digestion was conducted for 5
6 h with the maximum temperature not exceeding 140 C. Mineral
concentration (mg kg1) is expressed on a grain dry weight basis.
2.5. Statistical analysis
Data were analysed using the MINITAB 14 statistical package
using a General Linear Model analysis of variance with xed factors
[CO2] and TOS. Differences in the grain quality parameters between
[CO2] for each TOS were determined using the least signicant difference (LSD) at p 6 0.05 or as stated.
3. Results and discussion
3.1. Grain protein
In the 2008 growing season, grain protein concentration was
15.3% under [aCO2] and 13.4% at [eCO2] showing a 12.4% reduction
of grain protein concentration at [eCO2]. In 2009, grain protein concentration was 15.5% at [aCO2] and 13.5% at [eCO2], indicating an
almost similar reduction of grain protein at [eCO2]. Reduction in
grain protein concentration at [eCO2] has been reported elsewhere,
particularly under high temperate irrigated cropping systems
(Hgy et al., 2009; Kimball et al., 2001). However, the magnitude
of [eCO2] induced reduction in grain protein concentration was
higher in our experiment suggesting that [eCO2] is likely to have
a greater impact on grain protein levels under warmer and drier
conditions which commonly experienced in most of the Australian
wheat cropping systems. Higher reduction in grain protein concentration at [eCO2] was observed at TOS2 treatment than at TOS1 (Table 2). This is the rst study that investigated the interactive effects
of rising [CO2] and late sowing (high temperature and water stress

Table 1
Summary of climatic conditions. TOS1: sowing date according to local best practice, TOS2: late sowing date.

2008-TOS1
2008-TOS2
2009-TOS1
2009-TOS2

Rainfall during the

growing season (mm)

Growing degree days

(C) (during growth)

Days temperature 30
35 C during grain lling

Days
temperature > 35 C
during grain lling

TMAX during
grain lling (C)

TMAX during
growing season
(C)

Grain lling
duration
(days)

127
107
213
110

1372
1300
896
1755

4
4
2
3

3
4
3
12

25.5
26.6
26.2
35.5

19.0
20.2
20.3
22.8

43
37
67
19

Abbreviation: TMAX mean daily maximum temperature.

1309

N. Fernando et al. / Food Chemistry 133 (2012) 13071311

is common) on grain protein and mineral nutrient concentration

under a low yielding, dry-land wheat cropping system. In this
experiment, grain protein yield (g/m2) and grain N uptake (gm2)
were signicantly higher at [eCO2] (Table 2) suggesting that even
though plants take up more N at [eCO2], the nal result is that it
lowers grain protein concentration. The reduction in grain protein
concentration has been well documented at [eCO2] (Taub, Miller, &
Allen, 2008). However, the mechanism of reduction in grain protein concentration at [eCO2] is still not well known. There are various explanations and one of the common hypotheses is the
dilution of N due to the extra carbohydrate accumulation (Conroy,
1992; Gifford, Barrett, & Lutze, 2000). This explanation is highly
unlikely because biomass stimulation and reduction in grain protein concentrations are disproportionate (Taub et al., 2008). The
most feasible explanation for lower grain protein concentration
at [eCO2] is that there is a slow and reduced remobilization of N
to the grain during reproductive development at higher [CO2]
(Seneweera et al., 2005).

In both years, grain protein concentration was signicantly increased at TOS2 (17%, 16%) than at TOS1 (12%, 13%). On an average,
protein concentration increased by 40% and 17% at TOS2 in 2008
and 2009, respectively (Fig. 1). Such increases in protein concentration in TOS2 could be partly due to a lower grain yield which was
mainly due to higher temperatures and water stress experienced
in the later stages of plant development (Table 1). Increased grain
protein synthesis at high temperature (3035 C) has been documented elsewhere (Balla, Karsai, & Veisz, 2009; Panozzo & Eagles,
2000). In both years, TOS2 treatments plants experienced higher
temperatures (above 30 C) for 34 days during anthesis, which
may have accelerated grain protein synthesis. However, a smaller
increase of grain protein concentration in the 2009-TOS2 compared
to 2008-TOS2 could be related to the high temperature induced heat
shock (>40 C) experienced during grain lling (Balla et al., 2009). In
2009 TOS2 crops experienced > 35 C maximum temperature on the
3 days after anthesis including 7 days of >40 C maximum temperature during grain lling. In contrast, in the 2008-TOS2 growing

Table 2
Grain mineral concentration of Fe, Zn, S, Ca, grain yield and grain N uptake of Triticum aestivum L. cv. Yitpi grown under two [CO2] (ambient, 384 and 550 lmol mol1), two times
of sowing (normal, TOS1 and late, TOS2) in two growing seasons. Means and summary of two-way ANOVA results are shown.

Main effects
[aCO2]
[eCO2]
Percentage change
TOS1
TOS2
Percentage change
Interactive effects
TOS1
[aCO2]
[eCO2]
Percentage change
TOS2
[aCO2]
[eCO2]
Percentage change
ANOVA results
[CO2]
TOS
[CO2]  TOS

2008 Growing season

Fe (mg kg1)
Zn (mg kg1)

S (g kg1)

Ca (g kg1)

Grain yield (gm2)

Grain N uptake (gm2)

35.00
31.50
(10%)
26.63
39.88
(49%)

31.13
24.25
(22%)
24.38
31.00
(27%)

1.75
1.66
(5%)
1.50
1.90
(27%)

0.33
0.29
(15%)
0.27
0.35
(28%)

102
164
(61%)
152
114
(25%)

2.7
3.7
(37%)
3.1
3.2
(3%)

27.50
25.75
(6%)

27.25
21.50
(21%)

1.52
1.49
(2%)

0.29
0.26
(11%)

120
186
(55%)

2.6
3.7
(42%)

42.50
37.25
(12%)

35.00
27.00
(23%)

1.97
1.84
(7%)

0.38
0.32
(17%)

89
141
(58%)

2.7
3.8
(40%)

ns

ns

**

***

***

***

***

ns

ns (p = 0.057)
ns
ns

ns

ns

ns

ns
ns

41.10
36.80
(11%)
38.90
39.00
(0.3%)

26.00
22.90
(12%)
22.0
26.88
(22%)

1.83
1.65
(10%)
1.59
1.88
(19%)

0.37
0.34
(9%)
0.39
0.32
(16%)

150
223
(48%)
203
170
(16%)

4.0
5.2
(30%)
4.7
4.5
(4%)

40.00
37.75
(6%)

22.75
21.25
(7%)

1.83
1.65
(7%)

0.40
0.38
(5%)

178
227
(28%)

4.4
5.0
(14%)

42.75
35.75
(15%)

29.25
24.50
(16%)

1.59
1.88
(12%)

0.35
0.30
(13%)

122
218
(79%)

3.6
5.5
(52%)

***

2009 Growing season

Main effects
[aCO2]
[eCO2]
Percentage change
TOS1
TOS2
Percentage change
Interactive effects
TOS1
[aCO2]
[eCO2]
Percentage change
TOS2
[aCO2]
[eCO2]
Percentage change
ANOVA results
[CO2]
TOS
[CO2]  TOS

***

**

ns
ns

**

***

***

ns

ns

ns

ns

ns
ns (p = 0.09)

Abbreviations: ns, Not signicant (p P 0.05).

*
p < 0.05.
**
p < 0.01.
***
p < 0.001.

1310

N. Fernando et al. / Food Chemistry 133 (2012) 13071311

Fig. 1. (a) Grain protein concentration at elevated and ambient [CO2], (b) grain protein concentration at TOS1 and TOS2, (c) grain protein yield/m2 ground area at elevated and
ambient [CO2], and (d) grain protein yield/m2 at TOS1 and TOS2 in both growing seasons.

season, crops did not receive any days of temperatures > 40 C

throughout the cropping season. Balla et al. (2009) have demonstrated a similar response when they exposed wheat plants to
extremely high temperatures (>40 C temperature/heat shock) during grain lling and suggested that extreme temperature events
have a negative impact on grain protein synthesis.
3.2. Grain mineral concentrations
Grain Zn, Fe, Ca and S concentrations were lower under [eCO2]
in both growing seasons (Table 2). The overall reduction in grain Fe
and Zn concentrations was 10% and 22%, respectively for the 2008
growing season (Table 2). A similar trend in Zn and Fe reduction
was observed for the 2009 growing season. Similar reduction in
grain S (Erbs et al., 2010; Hgy et al., 2009) and Fe (Hgy et al.,
2009) concentrations have been reported for wheat grown under
[eCO2]. In this experiment, the reduction in grain Fe, Zn, Ca and S
concentrations under [eCO2] was more prominent at TOS2 (Table 2).
Large reductions of a range of macro and micro nutrient (Fe, Zn, Ca,
Cu, Mg, P and S) concentrations at [eCO2] have been reported in
controlled environment conditions (Seneweera et al., 1996).
Reduction of grain S (Erbs et al., 2010), Fe (Hgy et al., 2009) and
a decreasing trend of grain P, Mg, and Zn (Hgy et al., 2009) at
[eCO2] were reported in high rainfall, temperate wheat cropping
systems. However, it is still not known whether this reduction in
nutrients in grain is regulated through the suppression of nutrient
uptake or transport or by lower allocation to wheat grain at [eCO2].
Further, reduction in grain protein concentration was well correlated with S (r2 = 0.9), Fe (r2 = 0.7) and Zn (r2 = 0.5) suggesting that
a decline in grain protein concentration will follow similar reduction of these nutrients.
4. Conclusions
This is the rst report that showed that [eCO2] reduced wheat
grain protein, S, Zn and Fe under a low rainfall Mediterranean

environment, where the reduction in grain protein was higher than

previously reported in other FACE experiments. Grain nutrient concentration was increased in the delayed sowing treatment. However, the magnitude of reduction of these nutrients was highest
at [eCO2] in the delayed sowing treatment. Such a reduction in
the nutritive value of the wheat grain at [eCO2] is likely to have a
major impact on human nutrition. The strong positive correlation
between grain protein and S, Fe and Zn concentrations suggests
that any strategies developed to increase grain yield, will also have
to increase grain protein and micronutrient concentrations or at
least maintain them at present levels.

References
Ainsworth, E. A., & Rogers, A. (2007). The response of photosynthesis and stomatal
conductance to rising [CO2]: Mechanisms and environmental interactions. Plant
Cell and Environment, 30, 258270.
Balla, K., Karsai, I., & Veisz, O. (2009). Analysis of the quality of wheat varieties at
extremely high temperatures. Cereal Research Communications, 37, 1316.
Cakmak, I., Pfeiffer, W. H., & McClafferty, B. (2010). Biofortication of durum wheat
with zinc and iron. Cereal Chemistry, 87, 1020.
Carter, T. R., Jones, R. N., & Lu, X. (2007). New assessment methods and the
characterisation of future conditions. In M. L. Parry, O. F. Canziani, J. P. Palutikof,
P. J. van der Linden, & C. E. Hanson (Eds.), Climate change 2007: Impacts,
adaptation and vulnerability. Contribution of working group II to the fourth
assessment report of the intergovernmental panel on climate change (pp. 133
171). Cambridge, UK: IPCC, Cambridge University Press.
Conroy, J. P. (1992). Inuence of rising atmospheric CO2 concentrations and
temperature on growth, yield and grain quality of cereal crops. Australian
Journal of Plant Physiology, 21, 741758.
Corbellini, M. (1998). Effect of heat shock during grain lling on protein
composition and technological quality of wheat. Euphytica, 100, 147154.
Darroch, B. A., & Baker, R. J. (1990). Grain lling in three spring wheat genotypes:
Statistical analysis. Crop Science, 30, 525529.
Erbs, M., Manderscheid, R., Jansen, G., Seddig, S., Pacholski, A., & Weigel, H. J. (2010).
Effects of free-air CO2 enrichment and nitrogen supply on grain quality
parameters and elemental composition of wheat and barley grown in a crop
rotation. Agriculture, Ecosystems and Environment, 136, 5968.
Gifford, R. M., Barrett, D. J., & Lutze, J. L. (2000). The effects of elevated [CO2] on the
C:N and C:P mass ratios of plant tissues. Plant and Soil, 224, 114.
Hgy, P., Wieser, H., Kohler, P., Schwadorf, K., Breuer, J., Franzaring, J., et al. (2009).
Effects of elevated CO2 on grain yield and quality of wheat: Results from a 3year free-air CO2 enrichment experiment. Plant Biology, 11, 6069.

N. Fernando et al. / Food Chemistry 133 (2012) 13071311

Kimball, B. A., Morris, C. F., Pinter, P. J., Wall, G. W., Hunsaker, D. J., Adamsen, F. J.,
et al. (2001). Elevated CO2, drought and soil nitrogen effects on wheat grain
quality. New Phytologist, 150, 295303.
Loladze, I. (2002). Rising atmospheric CO2 and human nutrition: Toward globally
imbalanced plant stoichiometry? Trends in Ecology and Evolution, 17, 457461.
Mollah, M., Norton, R., & Huzzey, J. (2009). Australian grains free-air carbon dioxide
enrichment (AGFACE) facility: Design and performance. Crop and Pasture
Science, 60, 697707.
Panozzo, J. F., & Eagles, H. A. (2000). Cultivar and environmental effects on quality
characters in wheat. II. Protein. Australian Journal of Agricultural Research, 51,
629636.
Rogers, G. S., Gras, P. W., Batey, I. L., Milham, P. J., Payne, L., & Conroy, J. P. (1998).
The inuence of atmospheric CO2 concentration on the protein, starch and
mixing properties of wheat our. Australian Journal of Plant Physiology, 25,
387393.
Seneweera, S., Blakeney, A., Milham, P., Basra, A. S., Barlow, E. W. R., & Conoroy, J.
(1996). Inuence of rising atmospheric CO2 and phosphorus nutrition on the
grain yield and quality of rice (Oryza sativa cv. Jarrah). Cereal Chemistry, 73,
239243.

1311

Seneweera, S., Makino, A., Mae, A., & Basra, A. S. (2005). Response of rice to p(CO2)
enrichment: The relationship between photosynthesis and nitrogen
metabolism. Ecological Responses and Adaptations of Crops, 13, 3153.
Shewry, P. R., & Halford, N. G. (2002). Cereal seed storage proteins: Structures,
properties and role in grain utilization. Journal of Experimental Botany, 53,
947958.
Taub, D. R., Miller, B., & Allen, H. (2008). Effects of elevated [CO2] on the protein
concentration of food crops: A meta-analysis. Global Change Biology, 14,
565575.
WHO (2007). Preventing and controlling micronutrient deciencies in populations
effected by an emergency. Joint Statement by the World Health Organization,
The World Food Programme and the United Nations Childrens Fund. Online
citation: http://www.who.int/nutrition/publications/WHO_WFP_ ?tul=0?>UNICEFstatement.pdf.
Zarcinas, B. A., Cartwright, B., & Spencer, L. R. (1987). Nitric acid digestion and
multi-element analysis of plant, materials by inductively coupled plasma
spectrometry. Communications in Soil Science and Plant Analysis, 18,
131146.